Mammalian cell culture is widely used in the pharmaceutical and biotechnology industries for the manufacture of recombinant therapeutic proteins. The need to improve cell culture yield has increased tremendously in the last decade due to the growing market for protein therapeutics and an ongoing effort to improve production efficiency and to reduce the cost of goods manufactured. Chinese hamster ovary (CHO) cells are commonly used for the production of therapeutic proteins such as monoclonal antibodies, antigens and other specialized protein modalities.
The production of proteins using mammalian cells typically involves a fed-batch process, a process in which a nutrient supplement is fed to the cells throughout production that supports the cells' growth, metabolism and synthesis of a desired protein product. The current industry standard for cell culture fed-batch feeding processes is bolus feeding. In a bolus feeding process, nutrients are provided to the cells in intermittent discrete additions at various time points throughout the cell culture production. The bolus feeding process is simplistic in its approach, as it is confined by the practicality of manual feeding operation, which is one reason it is commonly employed.
Bolus feeding has several disadvantages, however, the foremost disadvantage being the inability to provide the precise nutrient quantities that the cells actually need. Stated mother way, bolus feeding is not tailored to the specific needs of the cell culture and consequently some nutrients may be provided in higher quantities than the cell culture requires, while other nutrients may be provided at levels less than those the cells require. Thus, while simplistic in methodology, the bolus feeding approach can lead to overfeeding, which consequently leads to overflow metabolism that results in an accumulation of waste byproducts, such as lactate, that are not supportive of cell growth or biosynthesis and may actually inhibit the growth of the cells.
Another disadvantage of a bolus feeding process in a manufacturing scenario is that bolus feeding processes have inherent sources of variability that may cause differences in cell culture performance. One such source is the variability in the timing of performing the feeding operation on a required feeding day. Yet another source of variability associated with bolus feeding is the rate at which a nutrient stream is administered into the bioreactor. Separately or together, variations in the time at which feeding is performed and the rate at which the cells are fed can affect the characteristics and production of a given cell culture from run to run. Still another disadvantage associated with bolus feeding is that it is typically a manual operation that needs to be performed by an operator. The lack of automation can consume human and financial resources and represents yet another source of variability, namely subtle differences introduced into a manufacturing process due to a lack of consistency between operators or, if the operator remains the same, uncontrollable operator-introduced variation.
The disadvantages of bolus feeding can be overcome through the use of a continuous feeding process. Continuous feeding processes can be designed to better meet cellular needs by continuously feeding smaller amounts of nutrients to the culture over time, rather than in large single bolus additions. In doing so, the nutrient concentrations can be controlled and maintained at more optimal levels for cell growth, thereby preventing overfeeding, minimizing the generation of unnecessary waste products and maintaining undisrupted pseudo-steady state levels. Employing a continuous feeding, operation can also eliminate the variability in the timing and the rate of feeding associated with a bolus feeding operation, since these variables are automation controlled in a continuous feeding process. Operator intervention is also eliminated by using a continuous feeding protocol.
While others have demonstrated different forms of this approach, such approaches still introduce the possibility of operator error. For example, Hu and Europa (U.S. Pat. No. 6,156,570) demonstrated a continuous feeding strategy that improved productivity. However, this continuous feeding strategy for mammalian cell cultures relies on equipment feedback control, which can introduce variability into a feeding process.
Summarily, a drawback common to all of these methods is the fact that they all rely on some sort of instrument-obtained feedback in order to manage the process. What is needed, therefore, is a method of feeding a cell culture that can be tailored to the specific needs of a given cell culture, can be automated and does not rely on instrument-mediated feedback to control the nutrients delivered to the cell culture.